The invention relates to the identification, quantitation and purification of insecticidal proteins from Bacillus thuringiensis, in particular from multigene strains of Bacillus thuringiensis.
Bacillus thuringiensis (hereinafter abbreviated Bt) is a gram-positive soil bacterium characterized by its ability to produce large crystalline parasporal inclusions during sporulation. These inclusions consist of proteins exhibiting a highly specific insecticidal activity (protoxins).
Many Bt strains with different insect host toxicity spectra have been identified. Numerous strains are active against larvae of certain members of the Lepidoptera (Bt strains active against over 100 species of lepidopterans have been identified to date), but strains showing toxicity against dipteran or coleopteran species are also known.
Bt parasporal inclusions have proven to be a valuable alternative to conventional insecticides. They are highly toxic to the target insects and harmless to the environment owing to their specificity. Other insect orders, animals and plants appear to be unaffected by the toxic crystal proteins. Various formulations of Bt have been used for more than two decades as biological insecticides to control pests in agriculture and forestry and, more recently, to control insect vectors of a variety of human and animal diseases.
Bt produces several types of toxins, the precise biochemical nature of which may vary from strain to strain. Some of them, in particular .alpha.- and .beta.-exotoxins, are toxic to a variety of insect orders or to many cell types. The parasporal crystal inclusion toxins (also called .delta.-endotoxins), which are proteins, have a more limited and specific host range. When consumed by a larva, Bt crystalline inclusions dissolve in the larval midgut and rapidly undergo proteolytical conversion into smaller toxic polypeptides (in the 23-80 kD molecular weight range) in the insect midgut. The produced toxin species interact with midgut epithelium cells of the host insect, generating pores in the cell membrane and disturbing the osmotic balance. The epithelium cells swell and lyse. The larva stops feeding and eventually dies. For several Bt toxins specific high-affinity binding sites have been demonstrated to exist on the midgut epithelium of susceptible insects, which could explain the extreme specificity of these toxins.
It has become clear in recent years that Bt is provided with a surprisingly large and variable family of insecticidal proteins. Data obtained using several experimental methods indicate that crystal protein genes in many subspecies of Bt that are toxic to lepidopterans are located on one or more large plasmids; in some subspecies, the gene may be located on the chromosome. The fact that the genes for the Bt protoxins are usually plasmid borne has made Bt a favourite candidate for genetic manipulations. This has resulted recently, among other things, in generating insect-resistant transgenic crop plants capable of expressing Bt crystal protein genes.
It has also become clear in recent years that many Bt strains contain several closely related genes coding for protoxins. For example Bt var. kurstaki NRD-12 strain is a three-gene strain. The presence of several such genes results in the production by a single Bt strain of several protoxins closely related by their amino acid sequences. Enzymatic action of proteolytic enzymes on such a mixture of protoxins, either in vitro or in the insect midgut, produces several toxins which may only differ by a few amino acid residues, which makes the obtained mixture of toxins difficult to separate. However, since these small differences frequently occur in key regions of the toxin sequence, they may result in significantly different toxicities towards a selected insect target. As the composition of the mixture of toxins produced by a multigene strain of Bt or the expression level of the individual toxins may vary with fermentation conditions, so may its relative toxicity to various insects, and, as a consequence, the host range of the multigene Bt strain. It becomes therefore important to monitor and quantitate different toxins present in endotoxin crystals, in order to optimize the production of the most wanted toxin from a multigene producer.
Various attempts to purify entomocidal toxins from .delta.-endotoxins of various strains of Bt are known from the prior art. The proposed methods typically include a digestion of crystalline inclusions of Bt with a proteolytic enzyme, such as trypsin, or insect digestive juices, followed by separation of the products of hydrolysis by various analytical procedures, such as electrophoresis, gel filtration and ion exchange chromatography. However, none of these methods demonstrated the ability to separate and purify closely related toxins obtained from a multigene strain of Bt.
Similarly, numerous methods of qualitative and quantitative characterization of Bt strains have been proposed. They involve, for example, the use of flagellar antibodies, probing for the genes with DNA probes or measuring the level of RNA production. These methods, although useful for characterization and classification purposes, are not satisfactory for quantitation of gene expression and monitoring the viability of a given strain or producing individual toxin standards for analyzing insecticidal activity, synergism, membrane studies or insect resistance.
More specifically in Fullmer, C. S. and Wasserman, R. H. Analytical Peptide Mapping by High Performance Liquid Chromatography. Application to intestinal calcium-binding proteins. J. Biol. Chem. 254, 7208-7212 (1979) a peptide mapping technique is described involving an exhaustive enzymatic digestion to provide a mixture of peptides. The analysis of the peptide mixture is carried out by reverse-phase HPLC, in association with an acid and an organic solvent.
In another reference, Yamamoto, T. Identification of Entomocidal Toxins of Bacillus thuringiensis by High Performance Liquid Chromatography. J. Gen. Microbiol. 129, 2595-2603 (1983), peptide mapping using the same Fullmer et al methodology is described. For example, see page 2601, line 5 under FIG. 4 where it is stated "A peak representing the proteinase resistant core could not be located". It is emphasized here that the use of organic solvent, either permanently reduces, or completely destroys, the biological activity of the toxins.
In a further reference, Yamamoto, T., Ehmann, A., Gonzalez, J. M. Jr. and Carlton, B. C. Expression of Three Genes Coding for 135-Kilodalton Entomocidal Proteins in Bacillus thuringiensis kurstaki. Current Microbiol. 17, 5-12 (1988), the same peptide mapping methodology is also followed. In this reference, the protoxin(s) is/are pre-purified, prior to trypsin hydrolysis. Following hydrolysis, the toxin mixture is denatured in urea and re-digested by trypsin for peptide mapping using reverse-phase HPLC (the peptides were solubilized in acid and eluted in acid/acetonitrile mixtures). As mentioned, above, this destroys the biological activity of the toxin. Unfortunately, the resultant peptide mapping, using single gene standards to interpret the results from multi-gene strains, failed to recognize the presence of the cryIA(c)[6.6] gene product in the strain kurstaki HD-1. This gene and its protein toxin is an important component, shown to be present by other researchers.
Further, in Hernstadt, C. and Wilcox E. Cloning and Expression of Bacillus thuringiensis Toxin gene Toxic to Beetles of the Order Coleoptera. U.S. Pat. No. 4,853,331 (1989) the cloning of a single gene and producing a single Bt protein by E. Coli host cells is described. The toxin is purified by affinity chromatography.
To the contrary, none of the aforementioned techniques will provide for the identification, quantitation and purification of protoxins, expressed by a Bt gene, while retaining the biological activity of the toxin. In fact, these references have a different purpose in mind, namely peptide mapping, wherein destruction of the biological activity of the toxin is irrelevant. However, in our invention, the biological activity of the toxin must remain substantially intact. Accordingly we cannot use the reverse-phase HPLC technique employed in these prior art techniques.